Our long-term objective is to understand fundamental molecular mechanisms of photoreceptor activation and cellular signaling in halobacterial phototaxis. In archaebacteria retinal-chromoproteins function as light energy converters and light signal transducers. Bacteriorhodopsin (bR) and halorhodopsin (hR) are light-driven proton and chloride pumps respectively, and the sensory rhodopsins I and II (sR-I,sR-II) are phototaxis receptors. Cells are attracted by orange-red light and repelled by UV-blue light, actively seeking an environment optimal for light absorption by bR and hR. These pigments undergo cyclic photoreactions in the millisecond (bR and hR) to second (sR's) range. A UV-absorbing intermediate of the sR-I photocycle, S(373), accumulates in natural light and functions as an additional photoreceptor. Upon photoactivation S(373) converts back to sR-I generating in the process a repellent signal. This unique photochromic system, sR-I/S(373), provides this primitive organism with a simple, yet effective, color discrimination system. Our aims are to identify the "activated" photoreceptor state(s) involved in signal generation, to characterize the molecular changes associated with the processes, and to establish the chemical or physical nature of the signal(s). Specifically we will: 1) isolate and spectroscopically characterize the sR-I/(373) system. 2) investigate its photochemical reactions and associated structural changes in the native membrane and in purified pigment in detergent micelles and in lipid bilayers. 3) identify signal- generating photochemical reactions. For this we will investigate the effect of retinal analogue substitution, double flash excitation, and mutagenesis on photocycle and phototaxis kinetics. 4) investigate the signaling mechanism(s). We will study the effect of components known to be involved in the signal transduction system on the photocycle kinetics and on the rotational diffusion of the photoreceptors and their thermal intermediates in both normal strains and in our newly isolated mutants with altered phototaxis. Absorption and laser-flash spectroscopy in the UV-visible and IR ranges, photoselection spectroscopy, resonance Raman scattering and measurements of intramolecular charge displacements will be used to gain insight on structural changes associated with photoreceptor activation.